# US 2013/0295136 A1 · Live attenuated catfish vaccine and method of making

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- Number: US 2013/0295136 A1
- Document ID: US20130295136A1
- Status: Published application
- Kind: A1
- Application number: 13/940,125
- Filed: 2013-07-11
- Published: 2013-11-07
- Earliest claimed: 2008-06-23
- Assignees: Mississippi State University
- Inventors: Mark L. Lawrence; Attila Karsi
- Web page: https://patentgrep.com/patent/US20130295136A1

## CPC

- A61K 39/025: {Enterobacteriales, e.g. Enterobacter}
- A61P 37/04: Immunostimulants
- C12N 15/102: {Mutagenizing nucleic acids}
- A61K 2039/522: {avirulent or attenuated}
- A61K 2039/552: {Veterinary vaccine}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A high throughput bioluminescence mutant screening procedure is disclosed. This procedure utilizes robotics, and bacterial luciferase to allow real-time monitoring of mutant viability. The procedure was used to decelop a live attenuated vaccine for a catfish against E. ictaluri, which is further claimed herein. Additionally, genes from other bacterial species are disclosed which may also be used to create vaccines.

## Claims

### Claim 1

A composition for providing immunological protection from diseases caused by Edwardsiella ictaluri which comprises a live attenuated strain of bacteria, wherein said live attenuated strain of bacteria is selected by introducing random transposon mutations into bacterial strains expressing bacterial luciferase operon and screening for said mutations by measuring reduction in bioluminescence.

### Claim 2

A composition for providing immunological protection from diseases caused by Edwardsiella ictaluri which comprises a live attenuated strain of the bacteria selected from the list of mutations consisting of: EiAKMut01, EiAKMut02, EiAKMut03, EiAKMut04, EiAKMut05, EiAKMut06, EiAKMut07, EiAKMut08, EiAKMut09, EiAKMut10, EiAKMut11, EiAKMut12, EiAKMut13, and EiAKMut14.

### Claim 3

A live attenuated vaccine prepared comprising a composition created by introducing random transposon mutations into bacterial strains expressing bacterial luciferase operon and screening for said mutations by measuring reduction in bioluminescence.

### Claim 4

A composition for providing immunological protection from diseases caused by Edwardsiella ictaluri which comprises a live attenuated strain of the bacteria having at least one mutation in the genes coding for glycine cleavage system (gcvP), serine hydroxymethyltransferase, succinate dehydrogenase, malate dehyrogenase, 2-oxoglutarate dehydrogenase, negative regulator of sigma E activity (rseB), hypothetical protein pEI1_p1, electron transport complex protein RnfB, Fimbrial chaperon protein, Putative RNA one modulator protein pEI1_p4, or UDP-glucose 6-dehyrogenase, fumarate reductase (frdA), and genes encoding enzymes in the tricarboxylic acid (TCA) cycle.

### Claim 5

A composition for providing immunological protection from diseases caused by bacteria which comprises a live attenuated strain of the bacteria having at least one mutation in the genes coding for glycine cleavage system (gcvP), serine hydroxymethyltransferase, succinate dehydrogenase, malate dehyrogenase, 2-oxoglutarate dehydrogenase, negative regulator of sigma E activity (rseB), hypothetical protein pEI1_p1, electron transport complex protein RnfB, Fimbrial chaperon protein, Putative RNA one modulator protein pEI1_p4, or UDP-glucose 6-dehyrogenase, fumarate reductase (frdA), and genes encoding enzymes in the tricarboxylic acid (TCA) cycle.

### Claim 6 (depends on 5)

The composition of claim 5 wherein the bacteria is of the genus Yersinia.

### Claim 7 (depends on 5)

The composition of claim 5 wherein the bacteria is of the genus Salmonella.

### Claim 8 (depends on 5)

The composition of claim 5 wherein the bacteria is of the genus Shigella.

### Claim 9 (depends on 5)

The composition of claim 5 wherein the bacteria is Escherichia coli.

### Claim 10 (depends on 5)

The composition of claim 5 wherein the bacteria is selected from the list consisting of Francisella tularensis, Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Haemophilus influenzae, Haemophilus ducreyi, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Actinobacillus actinomycetemcomitans, Avibacterium paragallinarum, Moraxella catarrhalis, Moraxella bovis, Pseudomonas aeruginosa, Coxiella burnetii, Bordetella bronchiseptica, Bordetella pertussis, Bordetella parapertussis, Bordetella avium, Burkholderia mallei, Burkholderia pseudomallei, Neisseria meningitidis, Neisseria gonorrhoeae, Brucella abortus, Legionella pneumophila, Helicobacteri pylori, Campylobacter jejuni, and Listeria monocytogenes.